利用核磁共振技術探討人類鹼性纖維母細胞生長因子與抑制血管新生藥物(SSR)的複合物結構

Study on complex structure between human basic fibroblast growth factor (hFGF-2) and anti-angiogenic drug SSR 〔2-amino-5-(1-(methoxy-2-methylindolizin-3-yl)methanone) benzoic acid〕by NMR

李長錦(Chang-Chin Li)

指導教授 ： 余靖

國立清華大學/理學院/化學系/碩士(2008年)

摘要

纖維母細胞生長因子是一群具有多樣生物活性的訊息傳遞分子;包括細胞成長與增生以及腫瘤的成長。其中FGF-2為一個具有肝素親和性的巨胜肽單體。許多研究已經顯示在正常細胞與病變細胞內均可發現 hFGF-2的存在，此外，當細胞進行分化及發展的過程中，FGF-2所調控的有絲分裂與血管新生具有關鍵的角色。特別的是FGF-2(18 kD hFGF-2)會以非傳統路徑進入細胞外介質並且利用與細胞表面的受器作用來啟動訊息傳遞。
最近血管新生抑制劑已經被證明為一種相當有潛力的抗癌藥物，此外，抑制纖維母生長因子的血管新生活性被認為是一個發展抗癌藥物的關鍵。在本篇論文中，我們探討一個有機化合物-SSR1281299E(簡稱SSR)藉由與FGF-2結合進而阻礙蛋白質與其受器(FGFR2)的交互作用，最後達到抑制血管新生及對抗腫瘤的效果。
首先，我們利用1H-15N HSQC擾動實驗來找出FGF-2與SSR的作用的胺基酸，進而描繪出結合位置。由穩定態變溫螢光和圓二色光譜的生物物理實驗發現SSR能夠穩定FGF-2蛋白質。此外，為了探討結合態的FGF-2構形，我們使用各種三維共振NMR實驗;包括HNCA、HNCO、CBCA(CO)NH、HBHA(CO)NH以及HCCH-TOCSY來完成結合態FGF-2內1H、15N 和13C的化學位移判定。在結構計算的部分，我們先由15N- edited與 13C- edited NOESY來得到蛋白質內所有的NOE資訊，並且整合氫鍵、雙面角及CSI等限制條件，最後利用ARIA-CNS軟體來解出在SSR結合下FGF-2的三維水溶液結構。
此外，為了解FGF-2與SSR結合位置的分子層次， 13C＆15N- filtered -13C edited NOESY技術被應用來得到複合物的分子內NOE資訊，接著配合HADDOCK軟體來對FGF-2蛋白質與SSR進行分子對應模擬，分析此複合物結構可幫助推測SSR抑制FGF-2活性的機制，而且此模型資訊有助於新一代藥物設計。

關鍵字

人類鹼性纖維母細胞生長因子 ； 生物核磁共振技術 ； 血管新生

並列摘要

The fibroblast growth factors (FGFs) are members of a large family of signaling molecules with many diverse biological activities that include cellular growth, proliferation and tumor growth. FGF-2 is a single-chain heparin-binding polypeptide and has been detected in normal and malignant cells and shown for its mitogenic and angiogenic activity with a consequent crucial role in cell differentiation and development. In particular, FGF-2 can be secreted to the extracellular medium where it switches on signal transduction pathways by binding to and activation of cell-surface receptors.
Angiogenic inhibitors recently have been demonstrated as potential anticancer agents. Moreover, inhibition FGFs angiogenic activity has been suggested as a crucial target for the development of anticancer agents. In this study, we describe an organic compound -SSR1281299E (abbreviate SSR) that binds to FGF-2 and inhibits the interaction between FGF-2 and its receptor, which is related to anti-angiogenesis and antitumor activity.
The binding region of SSR on the FGF-2 protein was characterized using 1H-15N HSQC perturbation experiments. Biophysical experiment of fluorescence and far UV CD (circular dichroism) suggested that SSR was able to stabilize hFGF-2. To determine the conformation of FGF-2 in the bound form, a variety of 3D NMR experiments include HNCA, HNCO, CBCA(CO)NH, HBHA(CO)NH and HCCH-TOCSY were used to accomplish the 1H,15N and 13C assignments in the bound form of FGF-2. After integrating all constraints that include 15N/13C- edited NOESY, dihedral angle, chemical shift index (CSI) and hydrogen bond, the 3D solution structure of hFGF-2 in complex with SSR was solved by ARIA-CNS. Moreover, To understand the molecular level binding site between FGF-2 and SSR, 13C＆15N-filtered-13C edited NOESY technique were applied to obtain the information of intermolecular NOE from complex system. By means of molecular modeling procedures, the structure-activity relationships between FGF-2 and SSR have been analyzed on the basis of the HADDOCK generated structures.

並列關鍵字

hFGF-2 ； BioNMR ； angiogenesis

參考文獻

2. Lobb, R. R. (1988). Eur J Clin Invest 18(4): 321-36.

3. Florkiewicz, R. Z. and A. Sommer (1989). Proc Natl Acad Sci U S A 86(11): 3978-81.

7. Caccia, P., G. Nitti, et al. (1992). Eur J Biochem 204(2): 649-55.

8. Thompson, S. A. and J. C. Fiddes (1991). Ann N Y Acad Sci 638: 78-88.

10. Seddon, A., M. Decker, et al. (1991). Ann N Y Acad Sci 638: 98-108.